Molecular mechanisms of DNA damage by PAHs. Polycyclic aromatic hydrocarbons (PAHs) can be a activated to reactive intermediates capable of inducing DNA damage by three major pathways. First, cytochromes P450 (P450) 1A1 and CYP1B1 can convert frans-dihydrodiols to (+)-a/tf/-diol-epoxides, which yield primarily a stable (+)-anft-fr"ans-N2-2'-deoxyguanosine (dGuo) adduct. Second, CYP peroxidase can convert the parent PAH to radical cations that form depurinating N7- and C8-guanine (Gua) and N7-adenine (Ade) adducts. Third, AKRs can convert trans-dihydrodiols to o-quinones, which redox cycle to generate reactive oxygen species (ROS). The ROS can directly modify DNA to form 7,8-dihydro-8-oxo-2'- deoxyguanosine (8-oxo-dGuo) as well as inducing the formation of lipid hydroperoxides. Lipid hydroperoxides undergo homolytic decomposition to form the bifunctional electrophile 4-oxo-2-nonenal, which then covalently modify DNA to form a heptanone-etheno-dGuo (HsdGuo)-adduct. We propose to use the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P) as a model PAH to explore the role of environmental exposure to PAHs in the induction of oxidative stress and formation of DNA-adducts. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for the specific analysis of (+)-anf/-frans-B[a]P-7,8,9,10-tetrahydro-7,8- diol-9,10-epoxide-N2-dGuo-adduct and quantified the formation of this adduct in four different human lung- derived cell lines. These studies have unexpectedly revealed that up-regulation of P450s 1A1 and 1B1 has a protective effect on DNA damage. We now propose to explore the molecular basis of this exciting new finding. Immunoaffinity stable isotope dilution LC-MRM/MS methodology has also recently revealed that lipid oxidation-derived HedGuo is present in the urine of subjects exposed to high levels of B[a]P through smoking. These studies will be extended to a B[a]P-exposed mouse model in which the B[a]P-derived urinary metabolites and DNA-adducts will be quantified together with urinary lipid oxidation products and HedGuo. Translational research will be conducted by monitoring the exposure of human populations to B[a]P through analysis of urinary B[a]P metabolites. The effects of this exposure will be assessed by monitoring urinary B[a]P-DNA-adducts, HedGuo, and oxidized lipids in the same subjects. We propose to address the following hypotheses: (1) P450 1A1 and 1B1 protect against DNA damage by facilitating removal of B[a]P metabolites through phase II enzymes. (2) Transport into the nucleus is an important determinant of DNA damage. (3) Analysis of oxidized lipids and B[a]P metabolites will define the relationship between oxidative stress and B[a]P exposure. (4) Quantitative analysis of urinary B[a]P- and a lipid oxidation-derived DNA-adducts will provide insight into the amount of DNA damage that has occurred in an animal model and in human populations exposed to B[a]P. The proposed research will be conducted under four specific aims. Aim 1. To analyze lipid oxidation- and B[a]P-derived DNA-adducts in cellular models of impaired nuclear transport. Aim 2. To develop stable isotope dilution LC-electron capture atmospheric pressure chemical ionization/MRM/MS and immunoaffinity LC-MRM/MS methodology for analysis of urinary B[a]P metabolites and B[a]P-derived DNA-adducts, respectively. Aim 3. To quantify urinary oxidized lipids, HedGuo, B[a]P metabolites, and B[a]P-derived DNA-adducts in a mouse model exposed to B[a]P. Aim 4. To quantify urinary oxidized lipids, HedGuo, B[a]P metabolites, and B[a]P-derived DNA-adducts in smoking and non-smoking human populations exposed to environmental B[a]P. Successful completion of the proposed research will permit provide a new approach and novel methodology for determining the inter-individual risk factors for DNA damage, which result from environmental exposure to B[a]P.